Calmodulin binding proteins were found to be substrates for protein-O-carboxylmethyltransferase (PCM; E.C. 2.1.1.24). Calmodulin, an acidic calcium binding protein was originally thought to be a major substrate for PCM. Kinetic analysis revealed that only 5% of calmodulin could be carboxylmethylated, and that its Km for PCM was = 350 MuM. Ca+2-dependent phosphodiesterase, calcineurin and Ca+2-calmodulin-dependent protein kinase were all carboxylmethylated with stoichiometric ratios of 0.2, 1.1, and 2.0 mol CH3/mol protein, respectively. Carboxylmethylation was found to reduce Ca+2-calmodulin dependent activities of each enzyme, with little effect on basal activities. Calcineurin, a Ca+2-calmodulin stimulated phosphatase, was the best substrate yet tested for PCM. These results suggest that PCM may regulate the calmodulin-stimulated activity of calcineurin, Ca+2-calmodulin protein kinase, and Ca+2-calmodulin-dependent phosphodiesterase.